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Journal Article
Research Support, Non-U.S. Gov't
Precise and efficient scarless genome editing in stem cells using CORRECT.
Nature Protocols 2017 Februrary
CRISPR/Cas9 is a promising tool for genome-editing DNA in cells with single-base-pair precision, which allows novel in vitro models of human disease to be generated-e.g., in pluripotent stem cells. However, the accuracy of intended sequence changes can be severely diminished by CRISPR/Cas9's propensity to re-edit previously modified loci, causing unwanted mutations (indels) alongside intended changes. Here we describe a genome-editing framework termed consecutive re-guide or re-Cas steps to erase CRISPR/Cas-blocked targets (CORRECT), which, by exploiting the use of highly efficacious CRISPR/Cas-blocking mutations in two rounds of genome editing, enables accurate, efficient and scarless introduction of specific base changes-for example, in human induced pluripotent (iPS) stem cells. This protocol outlines in detail how to implement either the re-Guide or re-Cas variants of CORRECT to generate scarlessly edited isogenic stem cell lines with intended monoallelic and biallelic sequence changes in ∼3 months.
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